The Gateway® Cloning System

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1 The Gateway® Cloning SystemCloning multiple fragments into a single vectorContentsHow to clone up to 4 DNA fragments simultaneously into one destination vector.Examples of expression of multiple genes in HeLa cells.Example of testing the effects of promoters and regulatory elements on protein expression.In the previous sections, we discussed the mechanism of transfer of a single DNA fragment among vectors via BP and LR recombination reactions. However, the full power of this system is realized when multiple DNA fragments are simultaneously assembled into a single vector in a predefined order, orientation, and reading frame.

3 Sample ApplicationsOptimized multigene delivery without co-transfectionExpression of enzymatic pathwaysExpression of multi-subunit protein complexesGene knock-down and rescue (controllable RNAi and heterologous gene expression from the same construct)Variable gene expression levels using different expression elementsCombinatorial taggingThe approach has multiple applications to the engineering of proteins, pathways, and cells, and provides a highly flexible platform for functional analysis.Invitrogen Proprietary & Confidential

4 More att sequences neededCTGCTTTTTTGTACAAACTTG attB1CAGCTTTCTTGTACAAAGTTG attB2CAACTTTATTATACAAAGTTG attB3CAACTTTTCTATACAAAGTTG attB4CAACTTTTGTATACAAAGTTG attB5StandardGateway®MultiSiteGateway®The recombination mechanism for creating a MultiSite Gateway® construct is the same as that with a single fragment. The difference is the att site specificity, which is determined at the single base level, as shown by the underlined nucleotides. Virtually no cross recombination is observed among them.Invitrogen Proprietary & Confidential

5 2-fragment MultiSite Gateway® ProPCR FragmentsattB1attB5rattB5attB2XXXXpDONRsattP1attP5rattP5attP2BP reactionsattL5attL2Entry ClonesXattL1attR5XIn MultiSite Gateway® and MultiSite Gateway® Pro, entry clones are also constructed via BP recombination but in order for them to have the correct configuration in the final LR assembly reaction, a combination of flanking attL and attR sites is used. This is facilitated by the modular nature of the att sites. A different set of pDONR vectors is required.In this example a 2-fragment recombination using MultiSite Gateway® Pro is shown. Here, by reversing the “standard”’ orientation of the attB5 site to an attB5r site in the PCR fragment, and by doing the same with its cognate attP5 counterpart in the donor vector, an attR5, instead of an attL5 is generated in one of the entry clones. The second entry clone bears a standard attL5 sequence that allows pairing with attR5 and the generation of an attB5 via LR recombination. This concept is key to the function of the MultiSite Gateway® system.To perform the LR reaction the entry vectors are mixed with an appropriate destination vector and LR Clonase™ II Plus. The reaction is incubated for 16 hours at room temperature and an aliquot is used to transform E. coli competent cells. Recombinants are selected using the destination vector’s antibiotic resistance.Destination VectorsattR1attR2LR reactionExpression clonesattB1attB5attB2Invitrogen Proprietary & Confidential

6 3-fragment MultiSite Gateway® ProPCR FragmentsattB1attB4attB4rattB3rattB3attB2XXXXXXpDONRsattP1attP4attP4rattP3rattP3attP2BP reactionsattL1attL4attL3attL2Entry clonesXXattR4attR3The same rationale is applied to a 3-fragment recombination scheme using MultiSite Gateway® Pro. However, a different arrangement of the att sequences and a different set of pDONR vectors is required as shown in this slide.Destination vectorattR1CmRccdBattR2LR reactionExpression cloneattB1attB4attB3attB2Invitrogen Proprietary & Confidential

9 Typical ResultsNumber ofExpected # colonies perTypical recombinationrecombining10 L reactionefficiency (%)fragments1103-10690-1002103-5801003103-10470-90Sufficient numbers of colonies with the expected expression construct are obtained using any of the described configurations.4102-10330-80Invitrogen Proprietary & Confidential

10 In silico cloning using Vector NTI AdvanceTM 10.3DNA of interestPrimers for PCR reactionCloning StrategyVector NTI Advance™ software allows the generation of Entry and Expression clones starting from any DNA sequence template and using any of the available configurations. It automatically designs the primers for the generation of the PCR fragments used in the corresponding BP reactions.The program is downloadable from the Vector NTI User Community at Licenses are free for academic and government researchers. Free 30-day trial licenses are available for commercial researchers by ingInvitrogen Proprietary & Confidential

11 Shortcomings when co-transfecting two plasmidsEGFPmRFPEGFP mRFPEGFPPCAGPlasmid 1This slide shows one of the shortcomings when co-transfecting two genes encoded by different plasmids. Some cells express one of the genes (EGFP) while others express only the other one (mRFP). In this particular example only one cell received and expressed both genes (marked with an arrow).MultiSite Gateway® overcomes this difficulty by recombining both genes into the same vector (see next slide)mRFPPlasmid 2Courtesy of Dr. Imamoto, Osaka University, JapanInvitrogen Proprietary & Confidential

12 Example: Expression of Multiple Genes in Human CellsCFPYFPApCMVB1YFPB4pEF1aB3CFPB2BB1pCMVB5YFPB4pEF1aB3CFPB2As mentioned in the previous slide, multiple genes can be combined into a single plasmid. Thus, only one transfection experiment needs to be performed. In this set of experiments, YFP and CFP are combined with the CMV and EF1alpha promoters. In one case, the CMV promoter is part of the destination vector (A), whereas in the other case the promoter is introduced using an entry clone (B).Transfection of only one plasmid was used in each of these examples, and virtually all cells exhibit expression of both genes.Invitrogen Proprietary & Confidential

13 Rapid Testing of Expression Elements using MultiSite Gateway®Kozak orPromoterIRESEGFPpABGHHeLaKozak orGtxDetermination of expression level of EGFPaurora A2xGtxcdc 25xGtxcyclin B112xGtxcyclin EEMCVmHCV2aIRES ( Internal Ribosome Entry Site )CMVmHCV33By swapping promoters and/or regulatory elements using MultiSite Gateway®, you can fine-tune gene expression.In this experiment, several promoters were tested for their effect on protein expression. Then, different Kozak or IRES elements were tested together with the CMV promoter. The plasmid constructs were transfected into HeLa cells, and the expression levels of eGFP were determined.EF1-amHCV45( CAG )HCV2a( SV40 )HCV33HCV45Courtesy of Dr. Imamoto, Osaka University, JapanInvitrogen Proprietary & Confidential

14 Rapid Testing of Expression Elements using MultiSite Gateway®Kozak orPromoterIRESEGFPpAHeLaTranscriptional signals with KozakTranslational signals with CMV promoter350713129940.05.010.015.020.025.030.035.040.0NoneKozakGtx2xGtx5xGtx12xGtxEMCVmHCV2amHCV33mHCV45300250Relative activity200150100These are the results from the experiment detailed in the previous slide. By swapping the promoter, dramatic changes in the protein expression levels were obtained. Fine tuning of the expression level is modulated by the use of different translation enhancers.50cdc 2CMVaurora Acyclin B1cyclin EEF1-aCourtesy of Dr. Imamoto, Osaka University, JapanInvitrogen Proprietary & Confidential

15 Summary for MultiSite Gateway® TechnologyMultiSite Gateway® Three-Fragment Vector Construction KitMultiSite Gateway® ProCompatible with…Ultimate™ ORF clonesattL1-attL2 entry clonesattR4-attR3 DEST vectorsMultiSite Gateway® Pro entry clonesattR1-attR2 DEST vectorsAvailable for…Only 3-fragment cloning2-, 3-, or 4-fragment cloningApplicationsVector constructionPromoter analysisExpression of multiple genes in one plasmidReporter analysis…and moreIn summary, Invitrogen offers two different configurations for the MultiSite Gateway® Technology.The MultiSite Gateway® three fragment construction kit is available for three-fragment cloning and is compatible with destination vectors that have attR4-R3 sequences.MultiSite Gateway® Pro is highly flexible in that you can use any standard Gateway® destination vector (attR1-attR2) for 2-, 3-, or 4-fragment cloning. With a wide range of Invitrogen’s destination vectors and those developed by your lab or collaborators, you are able to tap into a variety of applications. Some examples have been highlighted in this seminar, but the possibilities are endless.Invitrogen Proprietary & Confidential